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Dermatol Ther (Heidelb)
Dermatol Ther (Heidelb)
Dermatology and Therapy
2193-8210
2190-9172
Springer Healthcare Cheshire

39133361
1237
10.1007/s13555-024-01237-6
Original Research
Transungual Penetration and Antifungal Activity of Prescription and Over-the-Counter Topical Antifungals: Ex Vivo Comparison
Elabbasi Ali 1
Kadry Ahmed 1
http://orcid.org/0009-0000-6750-579X
Joseph Warren 2
http://orcid.org/0000-0002-1475-2874
Elewski Boni 3
http://orcid.org/0000-0002-8833-7323
Ghannoum Mahmoud mag3@case.edu

14
1 https://ror.org/051fd9666 grid.67105.35 0000 0001 2164 3847 Case Western Reserve University, Cleveland, OH USA
2 grid.260024.2 0000 0004 0627 4571 Arizona College of Podiatric Medicine, Midwestern University, Glendale, AZ USA
3 https://ror.org/008s83205 grid.265892.2 0000 0001 0634 4187 University of Alabama at Birmingham School of Medicine, Birmingham, AL USA
4 https://ror.org/0130jk839 grid.241104.2 0000 0004 0452 4020 Director, Center for Medical Mycology, University Hospitals Cleveland Medical Center, 11100 Euclid Avenue, Wearn 311, Cleveland, OH 44106-5028 USA
12 8 2024
12 8 2024
9 2024
14 9 24952507
16 5 2024
10 7 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc/4.0/.
Introduction

Topical antifungals for toenail onychomycosis must penetrate the nail to deliver an inhibitory concentration of free drug to the site of infection. In two ex vivo experiments, we tested the ability of topical antifungals to inhibit growth of Trichophyton rubrum and Trichophyton mentagrophytes, the most common causative fungi in toenail onychomycosis.

Methods

Seven topical antifungals were tested: three U.S. Food and Drug Administration-approved products indicated for onychomycosis (ciclopirox 8% lacquer; efinaconazole 10% solution; tavaborole 5% solution) and four over-the-counter (OTC) products for fungal infections (tolnaftate 1% and/or undecylenic acid 25% solutions). The ability to inhibit fungal growth was tested in the presence and absence of keratin. Products were applied either to human cadaverous nails or keratin-free cellulose disks prior to placement on an agar plate (radius: 85 mm) seeded with a clinical isolate of T. rubrum or T. mentagrophytes. After incubation, the zone of inhibition (ZI), defined as the radius of the area of no fungal growth, was recorded.

Results

In the nail penetration assay, average ZIs for efinaconazole (T. rubrum: 82.1 mm; T. mentagrophytes: 63.8 mm) were significantly greater than those for tavaborole (63.5 mm; 39.1 mm), ciclopirox (7.4 mm; 3.6 mm) and all OTC products (range: 10.5–34.2 mm against both species; all P < 0.001). In the cellulose disk diffusion assay, efinaconazole and tavaborole demonstrated maximal antifungal activity against both species (ZIs = 85 mm); average ZIs against T. rubrum and T. mentagrophytes were smaller for ciclopirox (59.0 and 55.7 mm, respectively) and OTC products (range: 31.2–57.8 mm and 25.7–47.7 mm, respectively).

Conclusions

Among all antifungals tested, the ability to penetrate human toenails to inhibit growth of both T. rubrum and T. mentagrophytes was greatest for efinaconazole, followed by tavaborole. These results indicate superior transungual penetration of efinaconazole compared to the other antifungals, suggesting lower keratin binding in the nail.

Keywords

Antifungal
Ciclopirox
Dermatophyte
Efinaconazole
Onychomycosis
Tavaborole
Tolnaftate
Topical
Undecylenic acid
http://dx.doi.org/10.13039/100019507 Ortho Dermatologics issue-copyright-statement© Springer Healthcare Ltd., part of Springer Nature 2024
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pmcKey Summary Points

Why carry out this study?	
Topical treatment for onychomycosis requires that antifungal agents deliver a sufficient concentration of drug through the keratin-rich nail to the site of infection.	
In the experiments reported here, we assessed the ability of seven topical antifungals—three U.S. Food and Drug Administration-approved products indicated for onychomycosis and four over-the-counter products used off-label for onychomycosis—to inhibit growth of the most common causative fungal species in toenail onychomycosis, either in the presence or absence of keratin.	
What was learned from the study?	
Among all antifungals tested, efinaconazole demonstrated the greatest ability to penetrate human toenails and inhibit the growth of both T. rubrum and T. mentagrophytes.	
Inherent antifungal activity of an antifungal agent is only one aspect in the treatment of fungal infections.	
In topical onychomycosis treatment, antifungals specifically formulated for greater nail penetration (e.g. with lower keratin binding) are more likely to deliver antifungal drug concentrations to the infection site.	

Introduction

Onychomycosis is a chronic fungal infection of the toenail bed or plate leading to thickening and discoloration of the nail, onycholysis, and alterations in skin surrounding the nail [1, 2]. The dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes are the most prevalent cause, accounting for over 70% of mycologically or histopathologically confirmed fungal toenail infections in North America [3, 4]. Elimination of the pathogenic fungus is the primary goal with onychomycosis treatment [5], although replacement of the diseased nail with healthy nail can take 12–18 months and rates of relapse and/or reinfection are high [2, 5].

Topical treatment of onychomycosis may be the preferred option among patients for whom systemic adverse events, drug-drug interactions, and contraindications associated with oral antifungals are of concern [2, 5, 6]. Moreover, while rates of microbiological resistance to oral antifungals are on the rise, there are no reports to date of naturally occurring dermatophyte resistance to topical antifungals [7]. Three topical antifungals have been approved by the United States Food and Drug Administration (FDA) for the treatment of toenail onychomycosis: ciclopirox 8% lacquer, efinaconazole 10% solution, and tavaborole 5% solution [8–10]. Numerous other topical antifungals (e.g., tolnaftate; undecylenic acid) are available as over the counter (OTC) products or sold in physicians’ offices; these products are marketed for the treatment of superficial fungal skin infections and used off-label for the treatment of onychomycosis.

Because dermatophyte infections in onychomycosis are generally found in the subungual space and nail bed, it is paramount that topical antifungal nail treatments are specifically designed to penetrate the nail and reach the site of infection at inhibitory concentrations. A major challenge to this is the highly keratinized nail plate itself, which provides a physical barrier to antifungal penetration [11]. Previous studies have investigated either the inherent inhibitory properties of ciclopirox, efinaconazole, and tavaborole in vivo or their ability to penetrate human cadaverous nails ex vivo [12–14]; in comparison, only one published study has assessed how ex vivo nail penetration impacts antifungal activity of ciclopirox versus tavaborole [15]. For antifungals available OTC, assays of human nail penetration are rare and typically predate the introduction of efinaconazole and tavaborole [16]. To our knowledge, a head-to-head comparison of efinaconazole, ciclopirox, and tavaborole along with OTC products has not been conducted. The objective of this ex vivo study was to directly compare the ability of the three FDA-approved and four OTC topical antifungals to inhibit growth of T. rubrum and T. mentagrophytes via penetration through cadaverous human nails versus diffusion through keratin-free cellulose disks.

Methods

Antifungal efficacy was tested for seven commercially available topical antifungals, of which three are FDA-approved for the treatment of onychomycosis (ciclopirox 8% lacquer; efinaconazole 10% solution; tavaborole 5% solution) and four are OTC products used off-label for onychomycosis (tolnaftate 1% and/or undecylenic acid 25% solutions [Formula 3, Formula 7, Terpenicol, and Tolcylen]; Table 1) [8–10, 17–25]. This study was performed with the approval of the Institutional Review Board at Case Western Reserve University. All donors or their families were required to sign a consent form specifying that their organs, eyes or tissues can be used for research. Table 1 Topical antifungal agents tested against Trichophyton rubrum and Trichophyton mentagrophytes

FDA U.S. Food and Drug Administration, OTC over the counter

aFor these experiments, ciclopirox was obtained from a commercial supplier (Sigma-Aldrich, Inc., St. Louis, MO, USA)

bCiclopirox is indicated for use as a component of a comprehensive management program, which includes removal of unattached, infected nails as frequently as monthly by a healthcare professional

cFour weeks recommended treatment duration for tinea corporis and/or tinea pedis; no recommended treatment duration for onychomycosis

dTwo weeks recommended treatment duration for tinea cruris; no recommended treatment duration for onychomycosis

All seven products were first assessed for their ability to penetrate human nails and inhibit growth of clinical isolates of T. rubrum (Mycology Reference Library [MRL] strains 34606 and 34607) and T. mentagrophytes (MRL strains 4439 and 42831). Cadaverous human great toenails were obtained prospectively from the National Disease Research Interchange (Philadelphia, PA); nails were considered acceptable if there was no malformation or diseased appearance, and no more than one nail was obtained from any donor. Each antifungal was evenly applied to the top of one cadaverous toenail and allowed to air dry for 30 min, after which five disks were punched from the middle of each nail using a biopsy punch (diameter: 4 mm) (Fig. 1); additional punches were also made from untreated nails. The thickness of each nail punch was measured using a digital caliper. Nail punches were then placed treated side up in the center of a 170-mm-diameter petri dish filled with potato dextrose agar and seeded with inoculum of one fungal strain (prepared to a final concentration of 2–5 × 105 conidia/mL). Because treated nail surfaces were not in contact with the fungal inoculum, any effects on fungal growth would require penetration of antifungal agents through the nail; untreated nails served as negative controls. After 2–7 days of incubation at 30 °C, the zone of inhibition (ZI)—defined as the radius of the area of no growth, disregarding any feathering—was measured. All antifungals and untreated nails were tested using five replicates against each fungal strain.Fig. 1 Methods for the nail penetration and disk diffusion assays. Schematics are for illustrative purposes and are not to scale. aZone of inhibition defined as the radius of the area of no fungal growth, disregarding any feathering

A second experiment assessed antifungal activity of all seven products in the absence of keratin. Each product was applied to a 6-mm-diameter keratin-free cellulose disk, allowed to dry, and placed in the middle of an agar plate seeded with 2–5 × 105 conidia/mL of one fungal strain (Fig. 1). Untreated cellulose disks served as negative controls. Plates were incubated at 30 °C for 2–7 days to allow for fungal growth prior to measurement of the ZI. Each antifungal was tested in triplicate against each fungal strain.

For each test product, mean (± standard deviation) ZI was calculated against each fungal species (ZIs were averaged across the two strains for each species). ZIs and thickness of nail disks were compared for efinaconazole versus each other product using two-tailed Tukey tests for multiple comparisons. P-values < 0.05 were considered statistically significant. Comparison of antifungal activity across the two experiments was determined by calculating mean nail penetration ZI as a percentage of mean disk diffusion ZI.

Results

Nail Penetration Assay

Among all products tested, antifungal activity via human nail penetration was greatest for efinaconazole. Among FDA-approved antifungals, ZIs against both T. rubrum and T. mentagrophytes were significantly larger for efinaconazole (82.1 and 63.8 mm, respectively) than for tavaborole (63.5 and 39.1 mm, respectively; both P < 0.001) and ciclopirox (7.4 and 3.6 mm, respectively; both P < 0.001; Fig. 2). For OTC antifungals, ZIs ranged from 12.5 to 34.2 mm against T. rubrum and 10.5–32.5 mm against T. mentagrophytes (Table 2). ZIs against both species were significantly smaller for all OTC antifungals than for efinaconazole (P < 0.001 for all comparisons). There was no inhibition of fungal growth with untreated control nails (all ZIs = 0 mm).Fig. 2 Antifungal activity in the nail penetration assay. Data are shown for U.S. Food and Drug Administration-approved topical antifungals indicated for onychomycosis. Bars represent mean (+ SD) zone of inhibition pooled from 5 replicates of each product against 2 strains of each fungal species (n = 10 replicates total per product). Zone of inhibition was defined as the radius of the area of no fungal growth, disregarding any feathering. Horizontal dashed line indicates maximum zone of inhibition (85 mm). ***P < 0.001 vs. efinaconazole. SD Standard deviation

Table 2 Antifungal activity of over-the-counter topical antifungals

Assaya	Species	Over-the-counter antifungalsb	Untreated	
Tolcylen	Formula 7	Formula 3	Terpenicol	
Nail penetration assay	T. rubrum	34.2 (7.2)***	32.3 (5.4)***	22.3 (4.3)***	12.5 (4.9)***	0 (0)***	
T. mentagrophytes	24.9 (2.8)***	32.5 (5.7)***	10.5 (1.9)***	17.8 (2.7)***	0 (0)***	
Disk diffusion assay	T. rubrum	42.2 (2.2)***	57.8 (12.6)***	43.7 (6.0)***	31.2 (16.7)***	0 (0)c	
T. mentagrophytes	32.3 (6.9)***	47.7 (9.1)***	25.7 (10.3)***	34.0 (3.8)***	0 (0)c	
Values in table are presented as the mean (standard deviation), in millimeters

***P < 0.001 vs efinaconazole

aIn both assays, the zone of inhibition, defined as the radius of the area of no fungal growth, disregarding any feathering, was measured

bTopical antifungals for the treatment of superficial fungal infections including tinea corporis, tinea cruris, and tinea pedis and used off-label for the treatment of onychomycosis

cNo statistical comparison of untreated to efinaconazole-treated disks due to lack of variance in both groups

Nail punches used to test efinaconazole against both strains of T. rubrum were significantly thinner than those used to test tavaborole (both P < 0.01; data not shown), but not different from those used to test ciclopirox. Nail punches used to test efinaconazole against T. mentagrophytes strain 42831 were significantly thicker than those used to test ciclopirox (P < 0.01) but not different from those used to test tavaborole. There were no differences in thickness of nail punches used to test these three drugs against T. mentagrophytes strain 4439. Nail punches used to test all OTC products against all fungal strains were either similar to or thinner than nail punches used to test efinaconazole.

Cellulose Disk Diffusion Assay

In the keratin-free disk diffusion assay, efinaconazole and tavaborole demonstrated maximal antifungal activity (ZIs = 85 mm) against both T. rubrum and T. mentagrophytes (Fig. 3). For ciclopirox, ZIs against both species (59.0 and 55.7 mm, respectively) were significantly smaller (P < 0.001 for both). ZIs for OTC antifungals ranged from 31.2 to 57.8 mm against T. rubrum and from 25.7 to 47.7 mm against T. mentagrophytes, and were significantly smaller than those for efinaconazole (P < 0.001 for all comparisons; Table 2). There was no inhibition of fungal growth with untreated control cellulose disks.Fig. 3 Antifungal activity in the cellulose disk diffusion assay. Data are shown for U.S. Food and Drug Administration-approved topical antifungals indicated for onychomycosis. Bars represent mean (+ SD) zone of inhibition pooled from 3 replicates of each product against 2 strains of each fungal species (n = 6 replicates total per product). Zone of inhibition was defined as the radius of the area of no fungal growth, disregarding any feathering. Horizontal dashed line indicates maximum zone of inhibition (85 mm). ***P < 0.001 vs. efinaconazole; no statistical comparison was made to untreated nails due to lack of variance in both groups. SD Standard deviation

To quantify the impact of nail penetration on the activity of FDA-approved antifungals, ZIs from the nail penetration assay were converted to a percentage of ZIs from the disk diffusion assay. Antifungal activity of efinaconazole against T. rubrum was nearly unaffected in the presence of human nails versus keratin-free cellulose disks, and approximately 75% of its antifungal activity against T. mentagrophytes was maintained (Fig. 4). In comparison, maintenance of antifungal activity against both species was lower for tavaborole and much lower for ciclopirox in the presence of human nails. Representative images of fungal inhibition with FDA-approved antifungals against one strain each of T. rubrum and T. mentagrophytes in both assays are shown in Fig. 5.Fig. 4 Maintenance of antifungal activity via nail penetration versus disk diffusion. Data are shown for U.S. Food and Drug Administration-approved topical antifungals indicated for onychomycosis. ZI was defined as the radius of the area of no fungal growth, disregarding any feathering. ZI Zone of inhibition

Fig. 5 Antifungal inhibition via cellulose disk diffusion and nail penetration. Representative images shown only for U.S. Food and Drug Administration-approved topical antifungals indicated for toenail onychomycosis. One of two strains tested for each fungal species is shown. Purple lines indicate zone of inhibition, defined as the radius of the area of no fungal growth, disregarding any feathering

Discussion

This study represents the first head-to-head comparison of human nail penetration and antifungal activity of the three topical antifungals currently approved by the FDA for onychomycosis treatment. Consistent with previous findings [15], tavaborole demonstrated greater antifungal activity than ciclopirox after penetrating through human cadaverous toenails. In comparison, efinaconazole demonstrated significantly greater antifungal activity than both tavaborole and ciclopirox as well as the four OTC topical antifungals evaluated (Formula 3, Formula 7, Terpenicol, Tolcylen). Although efinaconazole and tavaborole both demonstrated maximal antifungal activity when applied to keratin-free cellulose disks, this activity was maintained to a greater extent with efinaconazole than with either tavaborole or ciclopirox when required to penetrate through a human nail.

Physicochemical considerations to enhance the penetration of topical antifungals through the densely packed and highly keratinized cells of the nail plate include minimizing molecular weight, surface tension, and keratin binding [11, 13, 26–28]. Although efinaconazole is delivered in a vehicle that was chosen for its low surface tension [28], its molecular weight (384 g/mol) is considerably higher than that of ciclopirox (207 g/mol) and tavaborole (152 g/mol) [26]. Consistent with this, one study found that after the application to human cadaverous fingernails, the concentration of efinaconazole that penetrated into and through deeper nail layers was lower than that of either ciclopirox or tavaborole [29]. This may be offset, however, by differences in keratin binding, as efinaconazole has demonstrated lower keratin affinity and higher rates of release from keratin than ciclopirox and higher antifungal activity in the presence of keratin than both ciclopirox and tavaborole.[13, 14, 29, 30]. Efinaconazole has also demonstrated greater inherent in vitro antifungal potency; the minimum concentrations of efinaconazole required to inhibit ≥ 80% of T. rubrum or T. mentagrophytes fungal growth in vitro have been found to be dozens to hundreds of times lower than those for ciclopirox and tavaborole [31–35]. In the nail penetration assay, the significantly larger ZIs with efinaconazole compared to all other antifungals tested suggest a greater ability of efinaconazole 10% solution to deliver an inhibitory concentration of free drug through human nails.

Any investigation on the nail penetration and effectiveness of topical antifungals would ideally be performed in vivo with live patients with toenail onychomycosis. However, several practical considerations are inherently prohibitive to doing so, including the inability to accurately quantify or control for fungal load prior to or after application of antifungal agents, challenges to assessing nail thickness, prolonged treatment duration required to achieve antifungal effectiveness, and difficulty in identifying enough patients with infections caused by each of the four dermatophyte strains tested. Nevertheless, there are limitations to extrapolating findings from ex vivo penetration through disease-free cadaverous nail samples to the real-world treatment of onychomycosis. For example, although it has been suggested that healthy nails can be used to reliably estimate transungual penetration of diseased nails [27], onychomycosis-related changes to nail morphology may impact the ability of some antifungals to reach the infection site [36]. Moreover, morphological differences between healthy nails obtained from multiple donors cannot be completely controlled for in an ex vivo study; however, in the nail penetration assay, there was no apparent association between the thickness of treated nails and antifungal activity. While there was some variation in the thickness of nails used, these differences were not consistent, indicating that the differences in the ZIs observed cannot be reliably attributed to the thickness of nail punches used to test each drug. Additionally, antifungals were applied only once, whereas real-world use would involve weeks to months of daily treatment that may allow for the accumulation of higher drug concentrations at the infection site. Many dermatophyte species, including T. rubrum, can produce protective biofilms that sequester antifungal drugs and reduce their effective concentration at the site of infection [7]. Such biofilms are a characteristic feature of a particularly treatment-resistant form of onychomycosis called dermatophytoma [37]. In our nail penetration assay, the impact of biofilm formation on antifungal activity could not be assessed, although it is notable that efinaconazole has demonstrated greater efficacy than tavaborole in dermatophytoma treatment [37]. Finally, the unique application characteristics of the topical antifungals could not be modeled in this assay. For example, the low surface tension of efinaconazole 10% solution allows for access to the site of infection via application to the hyponychium and diffusion through the subungual space [38, 39], a route that cannot be exploited with topical lacquers. Because this route of access could not be tested in the nail-penetration assay, the present results may underestimate the antifungal activity of efinaconazole during real-world onychomycosis treatment.

Despite these limitations, the results are consistent with findings from clinical trials of prescription topical antifungals in the treatment of onychomycosis [8–10]. After 48 weeks of once-daily treatment, rates of mycological cure (negative fungal culture and negative KOH staining) were greater for efinaconazole 10% solution than for both tavaborole 5% solution and ciclopirox 8% lacquer + monthly debridement (53.4–55.2% vs 31.1–35.9% and 29–36%, respectively). Rates of complete cure, defined as mycological cure and complete clearance of target toenail, were two- to three-fold greater for efinaconazole (15.2–17.8% vs 6.5–9.1% and 5.5–8.5%, respectively). Unfortunately, drawing similar comparisons of efficacy with OTC products is challenging, as data from large, randomized clinical studies supporting their use in the treatment of onychomycosis are lacking [40, 41]. Because OTC antifungals are used off-label for onychomycosis, there is also a critical lack of guidance regarding treatment duration. Package inserts for OTC products state that treatment duration for superficial fungal skin infections is 2–4 weeks (Table 1), but the duration of onychomycosis treatment is significantly longer. Based on the relatively poor performance of OTC antifungals in the nail penetration assay, patients may experience a discouraging lack of efficacy within the first month of use and discontinue treatment altogether.

Conclusion

Although onychomycosis is a relatively benign condition, failure to effectively treat it can have severe consequences. Prolonged disease duration is associated with increased disease severity and greater treatment difficulty [42]. Untreated onychomycosis can spill over into widespread superficial fungal infection or become a far more dangerous invasive infection, as well as spread to other individuals [1, 43, 44]. Finally, suboptimal treatment patterns and poor treatment efficacy have contributed to the rise of antifungal resistance among dermatophytes, which threatens the spread of untreatable fungal infections [7]. It is therefore critical that the goal of onychomycosis treatment is the eradication of the causative fungus rather than just achievement of a normal-looking nail. In the present study, efinaconazole 10% solution had the greatest antifungal efficacy via transungual penetration among all antifungals tested, supporting its use for the topical treatment of onychomycosis.

Medical Writing/Editorial Assistance

Medical writing support was provided by Kevin Corcoran, PhD from Prescott Medical Communications Group, a Citrus Health Group, Inc., company (Chicago, IL), with financial support from Ortho Dermatologics. Ortho Dermatologics is a division of Bausch Health US, LLC.

Author Contributions

Ali Elabbasi, Ahmed Kadry, and Mahmoud Ghannoum designed and conducted this study and performed data analysis. Ali Elabbasi, Ahmed Kadry, Mahmoud Ghannoum, Warren Joseph, and Boni Elewski commented on previous versions of the manuscript and approved the final manuscript.

Funding

Sponsorship for this study and the Rapid Service Fee were funded by Ortho Dermatologics.

Data Availability

The datasets generated during this study are available from the corresponding author on reasonable request.

Declarations

Conflict of Interest

Ali Elabbasi and Ahmed Kadry have nothing to disclose. Warren Joseph has served as consultant and speaker for Ortho Dermatologics. Boni Elewski has provided clinical research support (research funding to University) for AbbVie, Anaptys-Bio, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Incyte, LEO Pharma, Lilly, Merck, Menlo, Novartis, Pfizer, Regeneron, Sun Pharma, Ortho Dermatologics, and Vanda; and served as a consultant (received honorarium) from Boehringer Ingelheim, Bristol Meyers Squibb, Celgene, LEO Pharma, Lilly, Menlo, Novartis, Pfizer, Sun Pharma, Ortho Dermatologics, and Verrica. Mahmoud Ghannoum has acted as a consultant or received contracts from Scynexis, Inc, Bausch & Lomb, Pfizer, and Mycovia.

Ethical Approval

This study was performed with the approval of the Institutional Review Board at Case Western Reserve University. The cadaver toenails were obtained by National Disease Research Interchange (NDRI), and all donors or their families were required to sign a consent form specifying that their organs, eyes or tissues can be used for research.

Prior publication/presentation: The data presented here have previously been presented in poster form at: Winter Clinical Dermatology Conference, 12–17 January 2024, Honolulu, HI, USA; ODAC Dermatology, Aesthetic, and Surgical Conference, 11–14 January 2024, Orlando, FL, USA; Maui Derm Hawaii, 22–26 January 2024, Maui, HI, USA; and Congress of Clinical Dermatology, 30 May-2 June 2024, Amelia Island, FL, USA. The poster presented at the Winter Clinical Dermatology Conference was published in the March 2024 supplement of Skin (Elabbasi A et al. Skin. 2024;8(2):s399).

Ali Elabbasi and Ahmed Kadry contributed equally to this work.
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